Chapter 3: Micropropagation of three Brachystelma species
3.2 Materials and methods
3.2.1 Explant decontamination and bulking up of experimental material
Stock plants of B. ngomense, B. pulchellum and B. pygmaeum were obtained (March and April, 2015) from the Botanical garden at the Univesity of KwaZulu- Natal, Pietermaritzburg where they were kept in a shade house. Voucher specimens (B. ngomense R. Br. A.
Shuttleworth 335 (NU), B. pulchellum R. Br. N. Hlophe 20 (NU) and B. pygmaeum R. Br. A.
Shuttleworth 322 (NU)) can be found at the UKZN Bews Herbarium (NU). Young stem and tuber explants were excised from the stock plants and thoroughly cleaned with running tap water. In the laboratory, the plants were further washed with liquid detergent followed by thorough rinsing with tap water. In the process of the surface decontamination treatment,
31 stem and tuber explants were submerged in 1% Benlate® (w/v) for 30 min followed by a solution of 70% ethanol (v/v) for 60 s. This process was accompanied with frequent agitation of the solution to ensure maximum contact of explants with the sterilant. The plant material was then rinsed three times, with distilled water. Thereafter, varying concentrations of two sterilants, namely sodium hypochlorite (NaOCl) (v/v) and mercuric chloride (HgCl2) (w/v) were used as independent treatments for the surface decontamination of the explants. The sterilant solutions were supplemented with a few drops of the surfactant, Tween 20. The plant material was kept in this solution for varying periods of time, during which the solutions were frequently agitated to allow for optimum contact between explants and solution. The explants were once again thoroughly rinsed, three times, with sterile distilled water under sterile conditions in a laminar flow bench. Surface decontaminated explants were further divided into lengths of ~10 mm each, and inoculated into culture tubes containing 10 ml of full strength MURASHIGE AND SKOOG (MS) (1962) basal medium supplemented with 30 g/L sucrose, 0.1 g/L myo-inositol and solidified with 8 g/L bacteriological agar. The agar was added after the pH of the medium was adjusted to 5.8 using either HCl or NaOH (Sigma- Aldrich) solutions. The medium was dispensed into culture tubes (100 mm x 25 mm, 40 mL) followed by autoclaving for 20 min at 121°C and 103 kPa. Sealed cultures were incubated in a growth room set at 25 ± 2°C under 16 h light/ 8 h dark photoperiod and PPF 40-50 µmol m-
2 s-1 provided by fluorescent (OSRAM) lamps. The number of explants for initial culture of each plant was approximately 50 depending on availability of plant material. After 4 weeks in culture, the number of sterile explants per treatment was recorded as a percentage. The in vitro-derived aseptic shoots obtained from the sterilization stage were continually sub- cultured until sufficient material was available to conduct subsequent experiments.
3.2.2 In vitro shoot proliferation
Upon obtaining sufficient plant material, an experiment was conducted to investigate the effects of N6-benzyladenine (BA), isopentenyladenine (iP) and meta-topolin riboside (mTR) on shoot proliferation and other shoot growth parameters. Full strength MS basal medium was used. The MS medium was supplemented with 30 g/L sucrose, 0.1g/L myo-inositol and different concentrations (1, 5, 10 and 25 µM) of BA, iP and mTR. The control for this experiment was MS basal medium without PGRs. Nodal explants were excised to a length of
~10 mm each, and inoculated into culture tubes. The cultures, 25 replicates per treatment, were incubated under controlled environmental conditions in a growth room set at 25 ± 2oC
32 and 16 h light / 8 h dark photoperiod and PPF 40-50 µmol m-2 s-1 provided by fluorescent lamps. Data on frequency of shoot proliferation, number of shoots per explant, length of longest shoot (mm), number of nodal segments, fresh weight (g), number of roots and root length (mm) were recorded after an incubation period of 6 weeks. This experiment was done simultaneously for all three species and was repeated.
3.2.3 In vitro rooting
This experiment was conducted to investigate the effect of the auxins, indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), on root induction and/or development. Surface decontaminated nodal explants of B. pygmaeum were excised and inoculated onto MS medium supplemented with 30 g/L sucrose, 0.1 g/L myo- inositol and varying concentrations (0.5, 5, 10, 15 and 25 µM) of IAA, NAA and 2,4-D. The control was MS medium without PGRs. The length of each explant was ~ 10 mm and tubes were used as culture vessels. The cultures, 25 replicates per treatment, were incubated under controlled environmental conditions in a growth room set at 25 ± 2oC and 16 h light / 8 h dark photoperiod and PPF 40-50 µmol m-2 s-1 provided by fluorescent lamps. Data on frequency of the number of roots and root length (mm) were recorded after an incubation period of 4 weeks. The period of incubation was based on “standard” tissue culture minimal incubation procedures. This experiment was performed only on B. pygmaeum based on its availability at the time. The experiment was repeated. A soil mix of 1:1 (v/v) vermiculite:sand was used for potting shoots that were transferred to ex vitro conditions. Prior to the vermiculite mixture, a soil mixture of 1:1 (v/v) rough sand: garden soil was tested but resulted in the death of all the plantlets.
3.2.4 Ex vitro rooting and acclimatization
The in vitro regenerated shoots (> 20 mm) were washed in water to remove any traces of agar. Ex vitro rooting was first investigated with the use of a 3 min pulse treatment using 492.1 µM indole-3-butyric acid (IBA). A second pulse treatment investigation was performed using 492.1 µM of IBA for 3, 12 and 21 min. After treatment with IBA, the shoots were potted in plastic planting trays (45 mm x 15 mm per well) containing 1:1 (v/v) vermiculite:sand mixture. The potted shoots were irrigated with ¼ strength 492.1 µM of IBA and incubated in a mist house in which high relative humidity (90-100%) was maintained using a high pressure fog system. The plantlets were kept in the mist house, during mid-
33 winter, under natural 12 h light / 12 dark photoperiod conditions for 3 weeks. Thereafter, the plantlets were transferred to a greenhouse with natural temperature (midday PPF of approximately 1000 µmol m-2 s-1) and natural photoperiodic conditions. The plantlets were watered with tap water every second or third day. Survival rate (%) was monitored once every week for a period of 5 weeks in the greenhouse. The period for which survival rate was monitored was completely dependent on how long the plantlets survived under natural conditions. The acclimatization procedure was similar for all experiments.
3.2.5 Effect of type of culture vessel on Brachystelma pygmaeum growth
Nodal explants were cultured onto full strength MS medium without PGRs in two types of culture vessels i.e. culture tubes (100 mm x 25 mm, 40 mL) and culture jars (110 mm x 55 mm, 300 mL). MS medium was prepared as stated in section 3.2.1. Single nodal explants were inoculated onto 10 mL (tubes) and 30 mL (jars) of the MS media. The cultures, twenty- five replicates per treatment, were incubated under controlled environmental conditions in a growth room set at 25 ± 2oC and 16 h light / 8 h dark photoperiod and PPF 40-50 µmol m-2 s-1 provided by fluorescent lamps. Results were recorded after 6 weeks in culture.
3.2.6 Effect of plant density
Nodal explants were cultured onto culture jars containing 30 mL of full strength MS medium without PGRs. The preparation of MS medium was as per section 3.2.1. The number of explants per jar was 1, 2, 3 or 4. The culture vessels were incubated under controlled environmental conditions in a growth room set at 25 ± 2oC and 16 h light / 8 h dark photoperiod and PPF 40-50 µmol m-2 s-1 provided by fluorescent lamps. Results were recorded after 6 weeks in culture.
3.2.7 Data analysis
A complete randomized experimental design was used for all experiments. Collected data were analyzed using one-way analysis of variance (ANOVA). Where there were statistical significances, mean values were further separated using Duncan’s multiple range test (DMRT) using SPSS for Windows (IBM SPSS Statistics 24, USA). Significant treatment effects were accepted at p ≤ 0.05. Reported values are mean ± standard error. Graphic representations were created using Sigma plot 8.0.
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